GSM1 Requires Hap4 for Expression and Plays a Role in Gluconeogenesis and Utilization of Nonfermentable Carbon Sources.

Multiple transcription factors in the budding yeast Saccharomyces cerevisiae are required for the switch from fermentative growth to respiratory growth. The Hap2/3/4/5 complex is a transcriptional activator that binds to CCAAT sequence elements in the promoters of many genes involved in the tricarboxylic acid cycle and oxidative phosphorylation and activates gene expression. Adr1 and Cat8 are required to activate the expression of genes involved in the glyoxylate cycle, gluconeogenesis, and utilization of nonfermentable carbon sources. Here, we characterize the regulation and function of the zinc cluster transcription factor Gsm1 using Western blotting and lacZ reporter-gene analysis. GSM1 is subject to glucose repression, and it requires a CCAAT sequence element for Hap2/3/4/5-dependent expression under glucose-derepression conditions. Genome-wide CHIP analyses revealed many potential targets. We analyzed 29 of them and found that FBP1, LPX1, PCK1, SFC1, and YAT1 require both Gsm1 and Hap4 for optimal expression. FBP1, PCK1, SFC1, and YAT1 play important roles in gluconeogenesis and utilization of two-carbon compounds, and they are known to be regulated by Cat8. GSM1 overexpression in cat8Δ mutant cells increases the expression of these target genes and suppresses growth defects in cat8Δ mutants on lactate medium. We propose that Gsm1 and Cat8 have shared functions in gluconeogenesis and utilization of nonfermentable carbon sources and that Cat8 is the primary regulator.

Protein Kinase A Negatively Regulates the Acetic Acid Stress Response in S. cerevisiae.

Bioethanol fermentation from lignocellulosic hydrolysates is negatively affected by the presence of acetic acid. The budding yeast S. cerevisiae adapts to acetic acid stress partly by activating the transcription factor, Haa1. Haa1 induces the expression of many genes, which are responsible for increased fitness in the presence of acetic acid. Here, we show that protein kinase A (PKA) is a negative regulator of Haa1-dependent gene expression under both basal and acetic acid stress conditions. Deletions of RAS2, encoding a positive regulator of PKA, and PDE2, encoding a negative regulator of PKA, lead to an increased and decreased expression of Haa1-regulated genes, respectively. Importantly, the deletion of HAA1 largely reverses the effects of ras2∆. Additionally, the expression of a dominant, hyperactive RAS2A18V19 mutant allele also reduces the expression of Haa1-regulated genes. We found that both pde2Δ and RAS2A18V19 reduce cell fitness in response to acetic acid stress, while ras2Δ increases cellular adaptation. There are three PKA catalytic subunits in yeast, encoded by TPK1, TPK2, and TPK3. We show that single mutations in TPK1 and TPK3 lead to the increased expression of Haa1-regulated genes, while tpk2Δ reduces their expression. Among tpk double mutations, tpk1Δ tpk3Δ greatly increases the expression of Haa1-regulated genes. We found that acetic acid stress in a tpk1Δ tpk3Δ double mutant induces a flocculation phenotype, which is reversed by haa1Δ. Our findings reveal PKA to be a negative regulator of the acetic acid stress response and may help engineer yeast strains with increased efficiency of bioethanol fermentation.

Architecture of androgen receptor pathways amplifying glucagon-like peptide-1 insulinotropic action in male pancreatic ß cells.

Male mice lacking the androgen receptor (AR) in pancreatic ß cells exhibit blunted glucose-stimulated insulin secretion (GSIS), leading to hyperglycemia. Testosterone activates an extranuclear AR in ß cells to amplify glucagon-like peptide-1 (GLP-1) insulinotropic action. Here, we examined the architecture of AR targets that regulate GLP-1 insulinotropic action in male ß cells. Testosterone cooperates with GLP-1 to enhance cAMP production at the plasma membrane and endosomes via: (1) increased mitochondrial production of CO2, activating the HCO3--sensitive soluble adenylate cyclase; and (2) increased Gαs recruitment to GLP-1 receptor and AR complexes, activating transmembrane adenylate cyclase. Additionally, testosterone enhances GSIS in human islets via a focal adhesion kinase/SRC/phosphatidylinositol 3-kinase/mammalian target of rapamycin complex 2 actin remodeling cascade. We describe the testosterone-stimulated AR interactome, transcriptome, proteome, and metabolome that contribute to these effects. This study identifies AR genomic and non-genomic actions that enhance GLP-1-stimulated insulin exocytosis in male ß cells.

SARS-CoV-2 infection of the pancreas promotes thrombofibrosis and is associated with new-onset diabetes.

Evidence suggests an association between severe acute respiratory syndrome-cornavirus-2 (SARS-CoV-2) infection and the occurrence of new-onset diabetes. We examined pancreatic expression of angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2), the cell entry factors for SARS-CoV-2, using publicly available single-cell RNA sequencing data sets, and pancreatic tissue from control male and female nonhuman primates (NHPs) and humans. We also examined SARS-CoV-2 immunolocalization in pancreatic cells of SARS-CoV-2-infected NHPs and patients who had died from coronavirus disease 2019 (COVID-19). We report expression of ACE2 in pancreatic islet, ductal, and endothelial cells in NHPs and humans. In pancreata from SARS-CoV-2-infected NHPs and COVID-19 patients, SARS-CoV-2 infected ductal, endothelial, and islet cells. These pancreata also exhibited generalized fibrosis associated with multiple vascular thrombi. Two out of 8 NHPs developed new-onset diabetes following SARS-CoV-2 infection. Two out of 5 COVID-19 patients exhibited new-onset diabetes at admission. These results suggest that SARS-CoV-2 infection of the pancreas may promote acute and especially chronic pancreatic dysfunction that could potentially lead to new-onset diabetes.

Mitochondrial Biogenesis Is Positively Regulated by Casein Kinase I Hrr25 Through Phosphorylation of Puf3 in Saccharomyces cerevisiae.

Mitochondrial biogenesis requires coordinated expression of genes encoding mitochondrial proteins, which in Saccharomyces cerevisiae is achieved in part via post-transcriptional control by the Pumilio RNA-binding domain protein Puf3 Puf3 binds to the 3'-UTR of many messenger RNAs (mRNAs) that encode mitochondrial proteins, regulating their turnover, translation, and/or mitochondrial targeting. Puf3 hyperphosphorylation correlates with increased mitochondrial biogenesis; however, the kinase responsible for Puf3 phosphorylation is unclear. Here, we show that the casein kinase I protein Hrr25 negatively regulates Puf3 by mediating its phosphorylation. An hrr25 mutation results in reduced phosphorylation of Puf3 in vivo and a puf3 deletion mutation reverses growth defects of hrr25 mutant cells grown on medium with a nonfermentable carbon source. We show that Hrr25 directly phosphorylates Puf3, and that the interaction between Puf3 and Hrr25 is mediated through the N-terminal domain of Puf3 and the kinase domain of Hrr25 We further found that an hrr25 mutation reduces GFP expression from GFP reporter constructs carrying the 3'-UTR of Puf3 targets. Downregulation of GFP expression due to an hrr25 mutation can be reversed either by puf3Δ or by mutations to the Puf3-binding sites in the 3'-UTR of the GFP reporter constructs. Together, our data indicate that Hrr25 is a positive regulator of mitochondrial biogenesis by phosphorylating Puf3 and inhibiting its function in downregulating target mRNAs encoding mitochondrial proteins.